A280

Measurement of absorbance at 280 nm is a well-established method for calculating the concentration of proteins in solution. The aromatic amino acids tyrosine and tryptophan absorb ultraviolet light, so the absorption coefficient of a protein can be inferred from its sequence. UV absorbance can also be affected by higher orders of protein structures, so A280 quantitation may be sensitive to pH and ionic strength. Alternative protein quantitation methods such as Bradford protein assay is available upon request.

Spectral analysis of Herceptin scanned between wavelengths 250 and 350 nm is shown. Concentration is determined by absorbtion at A280 and the ratio of A260 / A280 provides a quick method to determine if a contaminant is present, typical A260 / A280 ratio for purified protein is 0.6.

The Bradford Assay, a colorimetric protein assay, is based on an absorbance shift in the dye Coomassie when bound to arginine and hydrophobic amino acid residues present in protein. The (bound) form of the dye is blue and has an absorption spectrum maximum historically held to be at 595 nm. The anionic (unbound) forms are green and red. The increase of absorbance at 595 nm is proportional to the amount of bound dye, and thus to the amount (concentration) of protein present in the sample.

Octet Quantitation

Octet instruments perform label-free quantitation of antibodies and other recombinant proteins. Forte Bio's off the shelf biosensors include Protein A, Protein G, Protein L and Anti-His; all are compatible with our Octet HTX and 384 instruments. This approach provides fast and high throughput protein quantitation, including from culture supernatants. Octet titer determinations are useful for cell line development, process development, expression vector screening, construct design and protein engineering projects.

The graph below shows quantitation of an IgG1 antibody on an Octet instrument using Protein A biosensor tips. Comparison with a dilution series of an antibody standard at known concentrations allows the concentration of test samples to be determined.

HPLC

HPLC can be used to measure the concentration of a target protein in cell culture supernatant. Culture supernatants are loaded onto a column under conditions where the target protein binds, the column is washed and the target protein is then eluted (peaks in shaded area). Affinity resins such as protein A are used for measuring antibody concentrations; other columns such as ion exchange resins can be used for proteins lacking an affinity reagent. Absolute protein concentrations are determined by building a calibration curve to relate peak area to protein concentration using standards of known concentration. The figure shows a Herceptin standard curve from Protein A HPLC; large peaks are the bound load, the shoulder peaks are the wash and the peaks in the shaded region are elutions of target protein: pink peak is 1 mg/ml, blue peak is 0.5 mg/ml, yellow peak is 0.1 mg/ml and grey peak is 0.05 mg/ml of target protein (click on shaded region to zoom in).

ELISA (enzyme-linked immunosorbent assay) is a plate-based assay technique designed for detecting and quantifying substances such as peptides, proteins, antibodies, and hormones. In an ELISA, an antigen must be immobilized on a solid surface and then complexed with an antibody that is linked to an enzyme. Detection is accomplished by assessing the conjugated enzyme activity via incubation with a substrate to produce a measurable product. The most crucial element of the detection strategy is a highly specific antibody-antigen interaction. Many off the shelf ELISA kits are available that can be applied to determine the productivity of the protein being produced in our various expression systems or to support cell line development.

ATUM can provide quantitation of your protein by ELISA upon request.

SDS-PAGE:

Polyacrylamide gel electrophoresis separates the proteins in a sample based on molecular weight. Staining the gel with Coomasie stain allows an estimation of protein purity based on the number and intensity of bands observed.

HPLC

Proteins can be analyzed by reverse-phase chromatography. Quantitation is performed by integrating the area under the curves for the peak representing the target protein and its impurities.

The trace above shows an HPLC trace of Herceptin purified from HEK293 cells using a C4 column. Minor hydrophobic variant peaks are eluted after the main Herceptin peak.

Size Exclusion Chromatography

Size exclusion chromatography is suitable for separating and qualifying protein mixtures and is therefore a valuable technique for quality control in recombinant protein production. SEC separates solutes based on their size in solution rather than simply their molecular weight. Using appropriate protein standards, it is possible to construct a calibration curve by plotting retention time against size. The calibration curve allows the apparent size to be assigned to each peak.

Herceptin / HPLC / SEC Agilent

sdfr

Peak Retention Time (min)	Area %	SEC Calculated MW (kDa)	Peak Assignment
5.491	100	141.6	Product

Herceptin was purified using a single step affinity purification, desalted and run on HPLC-SEC. The calculated molecular weight of Herceptin based of its amino acid sequence is 145.3 kDa (2 light chains plus 2 heavy chains), consistent with SEC which assigns a molecular mass of 141.6 kDa to the single peak on the trace

HPLC / SEC Cytokine

Peak #	Peak Retention Time (min)	Area %	SEC Calculated MW (kDa)	Peak Assignment
1	5.60	2.40	144.9	Higher aggregates
2	7.921	93.87	12.2	Product
3	9.930	2.59	1.4	Impurity
4	10.984	1.14	0.5	Impurity

A cytokine was purified using a single step affinity purification, desalted and run on HPLC-SEC. Multiple peaks correspond to the product, higher aggregates and impurities.

HPLC / SEC PNGase F

Peak #	Peak Retention Time (min)	Area %	SEC Calculated MW (kDa)	Peak Assignment
1	5.731	4.65	70.54	Possible aggregate
2	6.785	4792.23	26.65	Product
3	8.830	120.02	5.5	Impurity

A peptide N-glycosidase (PNGase F) was purified using a single step affinity purification, desalted and run on HPLC-SEC. Multiple peaks correspond to the product, impurities and possibly higher aggregates.

Example Endotoxin Analysis Report

Bacterial Endotoxin Test is an in vitro assay for detection and quantitation of bacterial endotoxins, a component of the cell wall of gram-negative bacteria. This assay is also known as the Limulus Amebocyte Lysate (LAL) test and is sometimes referred to as the pyrogen test (because bacterial endotoxins can cause a fever in mammals, including humans). Endotoxin testing is provided as part of the analytical standard package for all proteins produced in mammalian cell culture at a scale of 1 Liter or greater. It can also be requested as a stand-alone test. Our LAL instrumentation loads the analysis report, shown above, into our LIMS system for review.

Octet

Octet instruments provide label-free kinetic analysis of binding interactions which can be used to assess affinities of antibodies. The ForteBio Octet accurately measures kinetic constants by bringing the detection surface directly to the sample. Unlike rough estimates of kinetic information from IC50 values obtained from ELISAs, real time kinetic measurements offer a direct and more realistic depiction of molecular interactions. The key advantages of measuring kinetics on the Octet system are easy and accurate determination of Ka and Kd for 96 or 384 samples with full kinetic profiles, and the ability to measure crude samples without purification.

The figure below shows an affinity screen with full kinetic characterization of Fab-ligand interaction. Antigen was covalently cross-linked to the Octet sensor via an amine linkage. Antigen coated sensors were placed into a Fab solution at a defined concentration for 300 seconds. Binding of the Fab to the antigen on the tip resulted in an increase in the Octet signal, which was proportional to the amount of Fab bound. The sensors were then moved to buffer for 300 seconds and the dissociation of the Fab from the antigen was measured by a decrease in Octet signal as the Fab fell off the tip. The ratio of dissociation rate (koff) to association rate (kon) gives an estimate of the dissociation constant (Kd) as a measure of antigen-antibody affinity.

Enzyme-Linked Immunosorbent Assay

ELISA (enzyme-linked immunosorbent assay) is a plate-based assay technique for detecting and quantifying substances such as peptides, proteins, antibodies, and hormones. In an ELISA, an antigen must be immobilized on a solid surface and then complexed with an antibody that is linked to an enzyme. The antibody-conjugated enzyme bound to the antigen is then incubated with a substrate; product formation then provides an indirect measure of antigen level. The most crucial element of this detection strategy is a highly specific antibody-antigen interaction.

ATUM can provide quantitation of your protein by ELISA upon request.

Reducing & Non-Reducing SDS-PAGE

SDS-PAGE and/or capillary electrophoresis (CE) is used to confirm the identify of molecules on the basis of molecular weight. The image below shows gels for 3 proteins: the monoclonal antibody Herceptin, a cytokine and PNGase F, each purified using a single step purification method. Data from PAGE gels or CE is converted to digital format and uploaded to our LIMS system. Digital data results are displayed and exported as virtual gels (shown).

SDS-PAGE gels show intact Herceptin running at 145.33 kDa non-reduced and shows 2 bands as expected, when reduced: a light chain band at 23.4 kD and a heavy chain band at 49.3 kD. Cytokine runs at 20.75 kD reduced, ∼22 kDa non-reduced. PNGase F runs at 35.73 kD reduced, ∼34 kDa reduced. The lighter, higher molecular weight bands with the PNGase may be hyperglycosylation variants.

Size Exclusion Chromatography

Size Exclusion Chromatography (SEC) separates molecules from largest to smallest in proportion to their molecular size in solution. Very large molecules are excluded from the packed bed and elute first while smaller molecules penetrate the pores of the resin to a degree that depends primarily on their size, causing them to elute later. Although most proteins are compact, some are more extended and have a larger than expected hydrodynamic radius leading them to elute earlier than expected. Other molecules may have charged sites that interact with the resin, resulting in an ‘ion-exchange’ effect that can delay when the protein is eluted, making it appear smaller than expected. Molecule-specific differences between molecular weight measured by SDS PAGE compared with the values from SEC can help to confirm identity.

Herceptin has a molecular weight of 145.3 kDa, in good agreement with HPLC-SEC data which assigns an apparent molecular weight of 141.6 kDa. In contrast, PNGase F sequence has a calculated molecular weight of 35.74 kDa (consistent with the apparent size on SDS-PAGE), while SEC shows an apparent molecular weight of 26.65 kDa indicating some interaction between the PNGase and the column. The Cytokine also appears to interact with the SEC column resin: its SEC-derived molecular weight is 12.2 kDa, compared with 20.75 kDa calculated from its sequence, and ∼22 kDa observed by SDS-PAGE. based off sequence which is predicted to be 12.2 kDa by HPLC-SEC. All predicted SEC masses were calculated using the retention time against a retention curve generated using known standards.

HPLC / SEC Herceptin

HPLC / SEC Cytokine

HPLC / SEC PNGase F

Herceptin / Western blot

Western blots are conducted by first running an SDS-PAGE gel and then transferring the proteins onto a membrane. An antibody specific to the target protein is then bound to the protein on the membrane which is then detected using a secondary antibody conjugated with an enzyme. This method can be used to identify the protein product or product related impurities.

A Western blot of reduced and non-reduced Herceptin probed with HRP conjugated anti-HuIgG1.

HPLC

HPLC can be used to assess heterogeneity within a sample and to determine the number of isoforms present. In the trace shown above, a Herceptin sample purified from HEK supernatant by a single step affinity process, was reduced with DTT and the resulting mixture (light and heavy chains) was analyzed using reverse-phase chromatography. The chromatogram shows a sharp peak corresponding to the light chain, and a more heterogeneous peak with resolved isoforms corresponding to the heavy chain.

Intact molecular weight

The 'intact mass' of a protein using mass spectrometry is its exact molecular weight. This analysis is a powerful way to confirm the identity of a molecule; it also quickly provides information on possible post-translational modifications. Intact mass analysis is most reliably performed on pure protein samples.

Deconvoluted mass spec of PNGase F under reducing conditions. Observed molecular weight is (MW) 35,933 daltons.

Antibody subunit analysis

When a protein is composed of more than one polypeptides as for example in antibodies, the masses of each polypeptide can be determined. In an antibody, the polypeptide chains are covalently held together by disulfide links which can be broken using a reducing agent. Subsequently, alkylation can prevent spontaneous re-formation of the disulfide bonds. The intact mass of each chain can be assessed using the TOF analyzers.

The spectra shown below are from mass spectroscopic analysis of Herceptin chains.

Deconvoluted mass spectrum of Herceptin heavy chain under reducing conditions. The observed molecular masses of (MW) 50,566 daltons, 50,366 daltons and 50,730 are consistent with different glycosylation modifications of the expected size of ∼49,120 daltons. To confirm, the sample was treated with PNGase F to remove N-linked glycosylations.

Deconvoluted mass spectrum of Herceptin heavy chain under reducing conditions after treatment with PNGase F. The three peaks observed in the un-treated sample (top) have collapsed into a single peak with the expected size 49,122 daltons.

Deconvoluted mass spectrum of Herceptin light chain under reducing conditions. The observed molecular mass of 23,433 daltons is consistent with the amino acid sequence.

Microchip capillary electrophoresis

Microchip based CE analysis has a resolution that allows determination of the relative abundance of the major N-glycan types found on antibodies, providing a high throughput option.

Labchip (µCE) Glycan analysis

N-glycosyation of proteins can be monitored by enzymatic N-glycan release followed by labeling with a fluorescent tag. Labelled glycans are separated using hydrophilic interaction chromatography (micro CE or HPLC) and detected by fluorescence. The chromatogram from µCE analysis (above) and HPLC (below) shows separation of labelled N-Glycans from Herceptin produced in HEK cells. The identities and relative abundance of each glycan is shown in the table below.

HPLC Glycan analysis

Labchip Charge Variant

Microchip based CE analysis allows identification of protein charge variants.

The profile of the monoclonal antibody shown below includes two basic variants, one main variant and three acidic variants which correspond to different post-translational modifications (PTMs). The resolution of the assay is sufficient to determine the relative abundance of each variant.