Quality Control Of Recombinant Antibody Drugs

Part1 Quality Control of Production Cells

The production cells of the recombinant monoclonal antibody should come from the same original cells, have the same genetic and biological characteristics, have no pathogenic microorganism contamination after comprehensive testing, and can stably and continuously express the correct structure and have biological activity under specific culture conditions. antibodies.

1 Structure Of Engineered Cells

First of all, the source and culture history of the cell line used should be clear and related background information, including cell species and geographical origin, pathogen detection results, initial isolation culture and line establishment information, methods and raw materials used, etc. In the construction, the means and steps of construction and screening, the sequence of the cloned gene, the coding region of the target gene inserted into the vector and related flanking sequences should be explained, the method of introducing the vector into the cell, the state and copy number of the vector in the cell, and the host should be provided. Genetic stability data after combining with the carrier. At the same time, the growth characteristics of the cells, culture conditions, composition of the culture medium, and the expression level of the introduced target gene should also be clarified.

2 Establishment Of Cell Bank

The establishment of cell banks can provide stable quality and continuous passage of cell seeds for recombinant antibody drugs, so as to ensure batch-to-batch consistency of antibody drug production. Similar to the requirements of other biotechnology drugs, the cell bank for antibody production is managed at three levels, namely the original cell bank, master cell bank and working cell bank. The original cell bank is a homogeneous cell population formed by the development of an original cell population into a stable cell population or through clonal selection.

The master cell bank and the working cell bank are respectively uniformly mixed by the original cell bank and the master cell bank after passage. Among them, the main cell bank shall not exceed 2 cell passages at most, and the working cell bank shall be limited at the same time. The subculture level of the cells during cryopreservation in the working bank must ensure that the amount of subcultured and proliferated cells after thawing can meet the production of a batch of products.

The subculture level of thawed cells should not exceed the highest limit passage approved for production. The management of the cell bank requires the establishment of ledgers, and each cell is clearly marked, including name, generation, batch number, and date of freezing. The main bank and the working bank should be stored separately, and the non-production cells should be strictly separated from the production cells. The cells of the above-mentioned seed banks at all levels should be used after passing the comprehensive inspection according to the specific requirements.

3 Cell Assay

The production cell verification of antibody drugs includes identification, pathogenic microorganisms, tumorigenicity and other inspections. At the same time, the recombinant antibody production cells constructed by genetic engineering technology should also include specific identification tests and cell matrix stability.

Cell Differentiation

Cell identification can be carried out by morphological, biochemical methods (isoenzyme, etc.), immunological detection (histocompatibility antigen, etc.), genetic detection (chromosomal karyotype, etc.), genetic marker detection (DNA fingerprinting, etc.), and other methods. Different methods should be used for joint detection. Antibody genes or their expression products can be identified by methods such as PCR, restriction endonuclease mapping, Southern hybridization, and Western blotting. After the establishment of the bank, a full inspection of the main bank and the final production cells should be carried out. At the same time, the cells of the newly built working bank should also be tested according to the specified requirements.

Pathogenic Microorganism Detection

The test includes sterility, mycoplasma, intracellular, and exogenous viral agents. The type and method of virus detection must be determined according to the species source and cell characteristics of the cells. Usually, viral factors can be detected by cell morphology observation, red blood cell adsorption test, different cell subculture methods, and inoculation of animals and chicken embryos. When detecting viral factors, a virus positive control and a hemadsorption positive control that can observe cytopathic changes should be established. For recombinant engineered cells, exogenous factor detection should also be performed on cell lysates or harvested fluids. For the detection of retroviruses and other endogenous viruses or viral nucleic acids, reverse transcriptase activity assays, transmission electron microscopy, and infectivity assays can be used. According to the species and tissue source of the cell line to be tested, the detection of special exogenous viral factors should also be carried out. For cell lines of mouse origin, hemorrhagic fever virus, lymphocytic choriomeningitis virus, reovirus, etc. should be detected; for cell lines of human origin, nasopharyngeal carcinoma virus, cytomegalovirus, hepatitis B and C viruses should be detected Wait.

Tumorigenicity Check

Tumorigenicity checks can be performed by in vivo assays in nude and neonatal mice, as well as by in vitro methods such as soft agar colony formation. At present, cell lines such as CHO produced by recombinant antibodies have been proved to be tumorigenic, and this inspection is usually no longer required. However, there are also opinions that recombinantly engineered cells inserted with specific gene sequences such as antibodies should be regarded as brand new cells and must be tested for tumorigenicity. Sexuality inspection, and experimental comparison during passage/amplification to observe changes in tumorigenic characteristics. Based on safety considerations, it is also necessary to optimize the purification process of antibody drugs and strictly control residual DNA and other components with potential tumorigenic risks during the release of the final product.

Cell Matrix Stability

As a recombinant antibody production cell containing a specific gene sequence obtained by DNA recombination technology, the producer must also have the stability data of the cell matrix, including the genetic stability of the recombinant cell, the stability of the expression of the target gene, and the stability of the continuous production of the target product , and the stability of the ability of the cells to produce antibody products during storage.

Part2 Quality Control Reference Material

Standard substances and detection methods are two important technical support points for the quality control of antibody drugs, and standard substances with stable and uniform properties are an indispensable and important factor in the establishment of methodology. The structure of biomacromolecules including antibodies determines their functions, so their quality control standard substances also include physical and chemical reference substances and active standards.

1 Preparation Of Standard Substances

The homogeneity and stability of the reference material is very important. Therefore, the raw materials need to undergo a comprehensive quality analysis, and even the standards on some key quality control items should be higher than those of the products. The selection of raw materials should follow the same principle as that of the test product, and its formula research should be based on the premise of not interfering with the measurement results, so as to improve the stability as much as possible. After comprehensive analysis and identification, physical and chemical reference substances are mainly used in routine quality control for confirmation of structure and post-translational modification.

Active standards are assigned values through collaborative calibration. The preparation method of ampule subpackage, freeze-drying and fusing sealing is the first choice for standard substance preparation because of its good airtightness. The dispensing of the standard product should be carried out in a clean environment. Under the premise of keeping the same conditions, samples should be taken at the same time interval before, during and after the dispensing to control the dispensing accuracy (≤1.0%). Sterility, moisture and other tests should also be carried out, usually the residual moisture of the reference material should be ≤3. 0%, but this indicator is related to stability, so the residual moisture in the standard substance should be reduced as much as possible. Antibody quality control standard substances are generally refrigerated or cryopreserved, and their validity period should be determined according to the stability evaluation. Stability studies include thermal acceleration tests, period checks and long-term stability tests.

The thermal acceleration test is to accelerate the chemical degradation or physical change of the sample under different temperature conditions, and predict the activity and content change of the standard substance at a given storage temperature through the analysis of thermodynamic parameters and mathematical models. Long-term testing refers to real-time stability monitoring under established storage conditions. In order to reflect the overall picture of the stability characteristics of the standard substance, multiple indicators should be set for evaluation, such as potency, purity, content, etc.

2 Physical And Chemical Reference Substances

The correct structure is the basis for recombinant antibodies to exert their biological functions. Physical and chemical reference substances are not only the premise and basis for establishing quality control standards for antibody drugs, but also simplify routine quality control methods, without involving large-scale analytical instruments such as mass spectrometry. Through comparative analysis between products and physical and chemical controls, it is possible to find out whether the product structure exists abnormal. The comprehensive identification of the structure of physical and chemical reference substances generally includes mass spectrometry relative molecular mass determination, terminal amino acid sequence determination, liquid mass peptide map or peptide fingerprint analysis, disulfide bond pairing mode, and glycosylation analysis representing post-translational modification, etc.

Mass Spectrometry Relative Molecular Mass Determination

The antibody is composed of 4 peptide chains, and the Fc segment has glycosylation modification, the relative molecular mass of the complete antibody can be directly measured, or the relative molecular mass of the antibody protein part can be determined after removing the sugar group with glycosidase, or by reducing After the disulfide bond is opened by the reagent, the relative molecular masses of the light and heavy chains of the antibody are determined respectively. At present, the determination of relative molecular mass by mass spectrometry mainly uses electrospray ionization (electrospray ionization, ESI) and matrix-assisted laser desorption ionization (matrix-assisted la-ser desorption ionization, MALDI) plasma source. After the protein is ionized, its mass-to-charge ratio is measured by a mass analyzer to calculate the relative molecular mass, and compared with the theoretical relative molecular mass to initially verify whether the antibody structure is normal.

Terminal Amino Acid Sequence Determination

Terminal amino acid sequence determination is also one of the ways to identify the primary structure of antibodies. Usually the N-terminal amino acid determination is most commonly used by the Edman degradation method. Due to the special structure of the antibody, the disulfide bond must be reduced first during sequencing, and the light and heavy chains must be separated by an appropriate method before determination. If the N-terminus of the antibody is blocked, it is necessary to use the corresponding method to remove the blocking type according to the blocking type, and then perform the determination according to the above procedure.

At present, the C-terminal amino acid sequence can be determined by enzymatic or chemical methods to collect C-terminal peptides, and then N-terminal sequencing; the collected C-terminal peptides can also be directly analyzed by tandem mass spectrometry. In addition, the relative molecular mass of the C-terminal peptides of different lengths obtained after being digested by carboxypeptidase can also be measured by mass spectrometer, and the C-terminal sequence can be determined according to the difference of adjacent relative molecular masses.

LC-MS Peptide Mapping Or Peptide Mass Fingerprint Analysis

In order to further identify the primary structure of the recombinant antibody, liquid mass peptide mapping or peptide mass fingerprint analysis can be performed. Liquid-mass peptide mapping is to hydrolyze antibodies into peptides with protease, and then detect them by mass spectrometry after chromatographic separation. The sequence of each peptide is determined by comprehensive analysis of relative molecular mass measurement results and protease hydrolysis characteristics. When the peptides obtained by enzymatic hydrolysis are directly detected by mass spectrometry, it is called peptide mass fingerprinting. The advantage of liquid-mass peptide mapping is that after the peptides are separated by liquid phase, the interference between each peptide is weak in the determination of the relative molecular mass of mass spectrometry, but it takes a long time; while the latter is suitable for high-throughput analysis.

Disulfide Bond Analysis

In IgG1 antibodies, there are 16 disulfide bonds in total, and the correct pairing of intra-chain and inter-chain disulfide bonds is an important guarantee for maintaining the spatial structure of recombinant antibodies and exerting biological functions. The disulfide bonds can be determined by liquid mass peptide mapping or peptide mass fingerprinting. That is, under non-reducing conditions, the recombinant antibody is directly digested with protease, and some peptides are still linked together by disulfide bonds after digestion. The disulfide bond pairing mode of the antibody can be verified by mass spectrometry identification of these peptides. In order to ensure the complete enzymatic digestion of the antibody, two or more proteases can be used for digestion if necessary, and the disulfide bond pairing of the antibody can be identified through comprehensive analysis.

Glycosylation Analysis

Sugar chains play an important role in maintaining the normal structure of antibodies and interacting with other proteins. The sugar chain modification of antibody Fc segment can significantly change its biological activity. Therefore, in the quality control of antibodies, the analysis of post-translational glycosylation modification is very necessary. At present, it mainly includes oligosaccharide map, glycosylation site analysis and sugar chain structure analysis. Oligosaccharide profiling is an analysis of the proportion of different modified oligosaccharide chains in recombinant antibody drugs. That is to say, after the glycosidase is derivatized and purified from the sugar chain cut off from the antibody, the proportion of each oligosaccharide chain is determined by liquid chromatography analysis. For further analysis of oligosaccharide diversity forms, literature data can be combined with corresponding oligosaccharide reference materials for comprehensive comparison. The glycosylation of antibodies mainly includes N-sugar and O-sugar.

N-acetylglucosamine (GlcNAc) at the reducing end of N-sugar and the amide nitrogen of asparagine (Asn) in the antibody form β1,4 glycoside The glycosylation site has a special sequence substructure “Asn-X-Ser/Thr”, where “X” is any amino acid except proline (Pro), and only the Asn in this sequence substructure Only then can N-glycosylation occur.

Although the O-sugar has no special sequence substructure, it can be linked to the hydroxyl group of serine (Ser) or threonine (Thr) in the antibody. The glycosylation site can be determined by the method of liquid mass peptide map or peptide mass fingerprint before and after the sugar chain is removed, and the glycosylation site can be determined by analyzing the difference in the relative molecular mass of the peptides in the map. There are currently many methods for sugar chain structure analysis: the sugar chain structure can be predicted by measuring the relative molecular mass of the sugar chain combined with existing literature; the sugar chain structure can also be determined by tandem mass spectrometry; or combined with a variety of glycosidases. Carry out step-by-step digestion of sugar chains to determine the connection mode of monosaccharides, etc.

3 Biological Activity Determination Standards

Activity determination is an important part of antibody quality control. Standards for biological activity determination also need to comply with the requirements of material uniformity and stable properties, and at the same time need to be accurately assigned. The preparation and stability evaluation of the activity standard for recombinant antibody quality control are the same as above. Usually, auxiliary materials such as stabilizers or excipients need to be added during the lyophilization preparation process, but the auxiliary materials can be added to the activity determination by increasing the amount of the active ingredient and increasing the pre-dilution factor before the activity measurement is performed by gradient dilution. interference to a minimum.

Although most of the current antibody biological activity determinations are based on the relative activity determined by comparing the EC50 value of the test product with the activity standard product, that is, no value is assigned to the standard product for biological activity determination, but if the test product, activity When the reduction rate of the standard biological activity is consistent with the trend, the relative biological activity expressed as a percentage cannot objectively reflect the change in the validity of the sample, so the assignment of the standard product can be used for reference in the way of other biotechnological drug activity standard products , that is, under certain experimental conditions, the weight unit corresponding to its EC50 value is defined as a biological activity unit, and the value is assigned through collaborative calibration research.

Collaborative calibration generally selects at least 3 laboratories to participate, and the invited units need to have relevant practical experience, excluding pre-tests, each unit should be able to provide at least 3 independent test results that meet the requirements and are carried out on different dates. Collaborative calibration should be carried out according to a unified method. The method should include specific operation steps such as sample reconstitution, dilution, and sample arrangement sequence. After calculation in a unified way, the results of the combined processing are combined with the help of statistical methods and the values ​​of the activity determination standards are assigned.

Part3 Quality Control of Recombinant Antibody Products

The quality control of recombinant antibody products is based on relevant laws and regulations, the requirements of guiding principles and the characteristics of the products themselves. Quality control of routine items of sex and injections.

1 Physical And Chemical Analysis

Structure Confirmation

In the routine quality control of antibody drug batch testing, the physical and chemical analysis of the structure mainly includes the determination of N-terminal amino acid sequence, peptide map, glycosylation analysis, etc., and is compared by introducing a fully analyzed physical and chemical reference substance. Therefore, in In the quality control of routine batch inspection, the workload of structure identification will be simplified. N-terminal amino acid determination isotropic control substance. In peptide map analysis, due to the complex and compact spatial structure of the antibody, the effect of protease hydrolysis is not ideal. Therefore, before protease hydrolysis, the antibody is generally denatured by chemical or physical methods, and then dithiothreitol Wait for the reducing agent to open the disulfide bond, and carry out alkylation protection on the reduced free sulfhydryl group. The enzymatic hydrolysis of the sample prepared under the same conditions as above and the physical and chemical reference substance will be more complete, and the subsequent HPLC separation will also obtain a more ideal peptide map. . In order to avoid the influence of sugar chains on the determination of peptide maps, it is sometimes necessary to cut them off before enzymatic digestion. Glycosylation analysis is based on the physical and chemical properties of the antibody drug itself, combined with physical and chemical reference substances and controlled by means of sialic acid content, oligosaccharide map analysis, etc. ratio is judged. Through the determination of N-terminal amino acid sequence, peptide map, glycosylation analysis, etc., combined with comparison with physical and chemical reference substances, it can effectively ensure the stability of the production process and the consistency of antibody drugs between batches.

Purity Analysis

The purity of antibody drugs directly reflects the level of purification technology and product quality. In order to avoid the deviation of one detection method in the detection of protein purity, generally at least two methods with different principles are selected for detection, so as to separate relevant impurities as much as possible and obtain relatively accurate purity results. Common methods for determining antibody purity include methods such as SDS-PAGE and capillary electrophoresis, as well as high-performance liquid chromatography such as reversed-phase, size exclusion, and ion exchange. Reduced SDS-PAGE electrophoresis is a more commonly used method, and the standard generally stipulates that the sum of the light and heavy chain band contents is ≥95. 0%. When liquid chromatography is used for purity determination, appropriate chromatographic columns and mobile phases should be selected to avoid antibody protein precipitation and hanging on the column during the analysis process; before formal analysis, the chromatographic column should be fully balanced and a blank control should be made, and there should be no miscellaneous peaks in the blank control appear; the area of the main peak is calculated by the area normalization method, and generally should not be less than 95% of the total area. 0%.

Determination Of Protein Content

The commonly used methods for protein content determination mainly include spectrophotometry, Lowry method, Bradford method, BCA method, Kjeldahl method, etc. Except for the Kjeldahl method, the principles of the other methods are related to the protein structure and amino acid composition, and the required reference substance for content determination must be consistent with the test substance. At present, most recombinant antibodies are assayed by spectrophotometry. Its extinction coefficient is based on the tryptophan, tyrosine, and cysteine model compounds established by Edel-hoch research. Under the condition of 6 mol L-1 guanidine hydrochloride, it can be used for other unfolded proteins under the same denaturing conditions. The extinction coefficient is estimated based on the principle. Combined with the denatured extinction coefficient, the extinction coefficient of the target protein can be obtained by correcting the absorbance values of different concentrations of natural proteins and denatured proteins, but the extinction coefficient is estimated through model compounds and comparative analysis with denatured proteins. At present, in the content determination of foreign biotechnology products, standard substances with the same structure as the test product have been used for content determination, but there is no public content traceability data. Kjeldahl nitrogen determination is used to determine protein content through nitrogen content analysis. The standard acid titration solution consumed in the reaction can be traced to the weight of anhydrous sodium carbonate with constant weight, so no other protein standard substances are needed, and it can be used as a method for traceability of content standards. At the same time, amino acid composition analysis is also one of the alternative means.

Analysis Of Other Physical And Chemical Properties

Such quality control mainly includes indicators such as relative molecular mass and isoelectric point. The relative molecular mass is also mainly determined by reducing SDS-PAGE electrophoresis, and the standard is generally stipulated to be within ±10% of the theoretical relative molecular mass, which is corrected by the mobility of the physical and chemical control. Isoelectric focusing electrophoresis is a common method for determining the isoelectric point of recombinant antibodies, and its pH gradient is mainly established by ampholytes. Since the presence of salt in the solvent disrupts the pH gradient, desalting can be done by dialysis, ultrafiltration, column passing, etc. When measuring the isoelectric point of a recombinant antibody, due to the uneven glycosylation of the antibody, the charge of the antibody is different, and multiple electrophoretic bands will appear in the measurement result. Therefore, the isoelectric point of the recombinant antibody is usually measured in a pH range. And it should be consistent with the physical and chemical reference substance that has been fully analyzed.

2 Biological Activity

Biological activity determination is an important quality control index to ensure the effectiveness of recombinant antibodies. After the antibody binds to its specific target, the therapeutic effect of the recombinant antibody is exerted through biological functions such as blocking cytokine signals, activating the complement system, and coupling toxin killing. The biological activity is mainly determined by establishing corresponding cell lines in vitro. Evaluate the model, simulate its mechanism of action to produce an objective full dose-effect response, and evaluate its biological activity by comparing with the active standard.

According to its mechanism of action, it is mainly divided into complement-dependent cytotoxicity, neutralization of killing activity or inhibition of cell proliferation after cell/growth factor signaling pathway blockade, reporter gene assay and other methods. If a specific cytological dose-response model cannot be established, the binding ability of the recombinant antibody to its target can also be determined as an evaluation index of its functionality. The main measurement methods include ELISA, flow cytometry and surface plasmon resonance technology. (surface plasmon resonance, SPR) biomolecular interaction analysis (biomolecular in-teraction analysis, BIA) and so on.

Biological Activity Determination of Recombinant Antibody

Complement Dependent Cytotoxicity (Complement Depend-ent Cytotoxicity, CDC) Method

This method was used to measure the activity of recombinant anti-CD20 monoclonal antibody. The recombinant antibody is serially diluted and then combined with effector cells expressing CD20. During the process of forming an antigen-antibody complex between the recombinant antibody and the cell surface antigen, the spatial structure of the antibody is changed and the complement-binding site of the Fc segment is exposed; Incubation and its cascade of activation completes the assembly of the membrane attack complex and punches the cell surface, ultimately leading to cell lysis. After the cells were stained, the antibody concentration was plotted against the absorbance, and the curve was fitted with a 4-parameter equation to obtain the “reverse S-shaped” curve of the whole reaction. The relative activity was calculated and evaluated.

Signaling Pathway Blockade

Agonists such as cytokines need to bind to receptors on the cell membrane and activate corresponding signaling pathways before they can exert their biological effects. The biological activity of recombinant antibodies targeting such cells/growth factors or their receptors can be based on this characteristic to establish corresponding biological activity assay methods, mainly including killing neutralization activity or specific cell proliferation inhibitory activity, etc. Taking anti-tumor necrosis factor-α (TNF-α) monoclonal antibody as an example, this recombinant monoclonal antibody can specifically recognize and bind TNF-α, and antagonize the cytotoxicity of TNF-α by blocking the binding of TNF-α to the receptor, so that It is protected from TNF-α-induced apoptosis. The antibody activity assay uses a cell line that is sensitive to TNF-α killing. The antibody is serially diluted and incubated with TNF-α to allow the antibody to bind to TNF-α. After the incubation, cells sensitive to TNF-α killing are added for culture, and the unneutralized TNF-α will bind to receptors on the cell membrane and induce apoptosis. The relative activity of the antibody can be evaluated by staining the surviving cells, measuring its absorbance value, and fitting the dose-effect relationship between the antibody concentration and the surviving cells with 4 parameters, calculating its EC50, and comparing it with the standard. The biological activity of the epidermal growth factor receptor (EGFR) monoclonal antibody is determined by the method of inhibiting cell proliferation, that is, by specifically binding and blocking the epidermal growth factor receptor, it can effectively inhibit the growth of tumor cells, and the test product and the standard product The dose-effect curve of proliferation inhibition was obtained by serial dilution, and the biological activity of the recombinant antibody was calculated.

Reporter Gene Method

The reporter gene method is also an activity evaluation method for determining the effect of signaling pathway changes after the recombinant antibody binds to the corresponding target. Generally, when the cell line is difficult to obtain, and the signal-to-noise ratio of the dose-effect curve produced by the killing neutralization or proliferation inhibition method is not ideal, based on the existing understanding of the pathway, the plasmid carrying the antibody target response element and the reporter gene can be combined After stably transfecting the cells, the biological activity of the recombinant antibody is detected by initiating the expression of the reporter gene through the interaction between the recombinant antibody and the corresponding target. For example, in the activity determination of VEGF monoclonal antibody, the plasmids carrying VEGF receptor and luciferase reporter genes were co-transfected into 293 cells, and cells stably carrying the above plasmids were obtained after screening. When VEGF binds to the receptor on the recombinant cell membrane, it can activate the expression of the luciferase reporter gene through the allosterism of the intracellular domain and the corresponding signaling pathway. The activity dose-effect curve of the VEGF monoclonal antibody can be obtained by co-cultivating with the recombinant cells after incubating the serially diluted test substance and the standard substance with a certain concentration of VEGF. Cell lines carrying similar reporter genes were also used for IL-1β monoclonal antibody activity assay.

Binding Activity Determination Of Recombinant Antibody

ELISA Method

Competition ELISA is the most commonly used assay for the binding activity of recombinant antibodies. Firstly, after the prepared soluble antigen is coated, the serially diluted test sample and standard product compete with a certain concentration of enzyme-labeled antibody to bind to the coated antigen, and the dose-effect relationship between antibody concentration and absorbance value is fitted by 4 parameters. , calculate the EC50 value of the test product and the standard product to evaluate the binding activity of the recombinant antibody.

flow cytometry

The principle of this method is the same as that of competition ELISA, except that the coated soluble antigen or receptor is replaced by cells with specific binding sites or membrane surface marker molecules, and the effect of competitive binding is detected by measuring the positive cell rate by flow cytometry. This method avoids the preparation process of the corresponding antigen, and it can be used to measure the binding activity of recombinant antibodies that are difficult to obtain soluble antigens. At the same time, the spatial structure of cell surface antigens is close to the naturally occurring form, and the measurement results should be more objective.

BIA Method

The SPR-based BIA method has a short measurement period and less consumption of samples to be tested. This method does not require labeling when measuring antigen-antibody binding activity, avoids the influence of labels on molecular structures, and can more truly reflect the binding situation of antigen-antibody. Its detection chip can immobilize soluble antigens and cells. When the antibody to be tested flows through the chip through the microfluidic flow system, once it binds to the immobilized antigen, the protein concentration on the surface of the chip will increase, which in turn will cause a change in the refractive index of the liquid on the surface of the chip and be detected. detected. The advantage of BIA technology is that it can dynamically monitor the binding reaction of antigen and antibody in real time, and determine the reaction parameters such as affinity constant.

3 Residual Impurities

Recombinant antibodies are generally expressed and produced by mammalian cells, so it is necessary to limit the amount of exogenous components that may exist in the product from the expression system and purification process. According to its production process, the limit control of related impurities mainly includes residual DNA, residual host cell protein, residual protein A, etc. Among them, the detection of the residue limit of host cell protein and protein A (the ligand of the affinity purification column) mainly adopts the method of sandwich ELISA. The DNA of passaged cell lines theoretically has the potential risk of tumorigenicity. The FDA stipulates that the limit of residual DNA allowed by biological products is no more than 100 pg per dose. my country also stipulates that sensitive methods must be used to detect DNA derived from host cells (including transformation). The residual DNA content of passaged mammalian cells) is limited to less than 100 pg per dose. At present, the detection methods of residual DNA mainly include methods such as hybridization, fluorescent staining, and quantitative PCR. The hybridization method mainly uses non-radioactive materials such as digoxin to label nucleic acid probes, and does not require special equipment. However, in actual work, it is usually necessary to perform simultaneous processing such as protease digestion on the test sample and DNA standards, which will reduce the detection sensitivity. The fluorescent dye method uses fluorescent dyes such as PicoGreen that can specifically form complexes with double-stranded DNA, and detects with a fluorescent microplate reader. According to the standard curve and the fluorescence intensity of the test product, the amount of DNA residue is calculated. This method is simple and quick, but due to the high quality control standard and detection limit of residual DNA in antibody drugs, fluorescent staining is usually not suitable for the control of DNA residues in antibody drugs.

4 Finished Product Quality Control Of Recombinant Antibody Drugs

In addition to testing the content, biological activity and necessary physical and chemical properties of the finished recombinant antibody drug, it should also be tested for other routine items in accordance with the requirements of injections, including identification test, appearance, visible foreign matter, content, moisture, pH value, Sterility inspection, bacterial endotoxin inspection, abnormal toxicity inspection, etc. For specific determination methods, please refer to the current Pharmacopoeia of the People’s Republic of China and other relevant technical documents.

Part4 Recombinant Antibody Drugs Need To Be Carried Out In The Future

The quality control technology of recombinant antibody drugs in my country has been continuously developed and improved with the deepening of antibody drug research and development. In the future, the quality control of antibody drugs still needs to pay more attention to the following main aspects.

1 Quality Control Of Production Cells

The current version of the Pharmacopoeia’s requirements for cell bank management and verification have been proved by years of practice to ensure the overall quality of recombinant antibody and other biotechnological drug production cell lines, but the reverse transcriptase activity test, which is a common method for retrovirus detection, still needs to be further improved. , that is, it is necessary to establish a uniform and stable RNA template and a reverse transcriptase activity standard, and use methods such as quantitative PCR to quantify the enzyme activity.

2 Structural Analysis Of Recombinant Antibody

The application of new instruments such as mass spectrometers, capillary electrophoresis instruments, and new high-performance liquid phase systems can effectively improve the level of structural analysis in antibody quality control. For example, on the basis of sufficient research and identification of recombinant antibody physical and chemical reference substances, foreign research and development units have raised the quality control standard from qualitative analysis of post-translational repair peptides to quantitative detection in the quality control of recombinant antibody peptide maps.

Restricted by various conditions, the current research on physical and chemical reference substances for quality control of recombinant antibody drugs in my country is not exhaustive enough, and the results of peptide map analysis in the quality control standards are only required to be consistent with the reference substances. At the same time, the post-translationally modified sugar chains of recombinant antibodies are very complex, and their structures are related to biological functions. Therefore, glycosylation analysis is very challenging, and therefore it is also the focus and difficulty of antibody quality control. At present, although mass spectrometry and other analysis methods can be used to analyze sialic acid, oligosaccharide maps and glycosylation sites, and reflect certain glycosylation information, there is currently no simple and easy method that can quickly analyze sugar chains. The structure was fully analyzed and identified.

3 Detection Of Biological Activity Of Recombinant Antibody

Antibody activity detection methods are diverse due to their different functions. Usually, cell evaluation models are used in vitro to simulate their mechanism of action to produce dose-effect responses. However, in actual work, some activity detection cycles are longer, the steps are cumbersome, and there are many influencing factors. , the reproducibility is not ideal, and some recombinant antibody drugs cannot find a suitable cell line for activity determination, and then use antibody binding activity as its functional evaluation index. For the above links that need to be further improved, corresponding transgenic cells can be constructed based on the understanding of intracellular signal transduction pathways, in order to establish a more accurate, durable and convenient activity determination method. On the other hand, the current standards for the determination of antibody biological activity are mainly reflected in the percentage relative to the activity of the standard product, which is a result of relative biological activity. If we can learn from the quality control experience of other biological technology drugs, the recombinant antibody Scientifically assigning and defining the biologically active units of drugs, and strengthening the research on the stability of standard substances, will be able to evaluate the effectiveness of recombinant antibody products more objectively and effectively, and will also facilitate comparative research between similar antibody products.

4 Residual Impurity Analysis Of Recombinant Antibody

In the quality control of recombinant antibodies, limited control of host cell protein and protein A residues is required. Although CHO cells and protein A are mainly used in antibody production, the cell lines and production processes of each unit are not consistent. Therefore, the research and development unit should prepare corresponding residual control substances and antibodies according to their respective production processes, and establish corresponding detection methods. If a commercial kit is used, its specificity, detection limit, recovery rate and other parameters must be verified before it can be used.

Due to the large dosage of recombinant antibody drugs, although the hybridization method and fluorescent staining method can control the residual amount of nucleic acid in the product by increasing the recovery rate of the standard and enriching the nucleic acid in the sample to be tested, etc., due to the limitations of the method Still need to be further improved. The quantitative PCR method has unique advantages in terms of specificity, sensitivity, and accuracy, and can objectively quantify residual DNA and reflect the relative molecular mass distribution of fragments. However, this method still needs further optimization and standardization of primer design and standard products. At the same time, the detection of residual DNA also needs to be combined with process verification for comprehensive control.

Part5 Conclusion

How to control the quality of recombinant antibody drugs more scientifically and effectively requires further in-depth research on quality control methodology and related reference materials in combination with clinical evaluation and post-marketing safety monitoring. This article summarizes the research progress and problems to be solved urgently on the quality control of recombinant antibody drugs at home and abroad, with the aim of attracting more attention and discussion on related issues.